Compound-conjugated pokeweed antiviral protein (PAP) and conjugates of other natural toxins, such as ricin and diphtheria toxin, have long held the promise of therapeutic efficacy. In theory, the presence of a natural ligand as the “compound” portion of the conjugates results in target cell damage, and no other cellular damage. In practice, imprecise targeting results in toxicity, due, in part, to unconjugated toxin causing unintended cellular damage. With regard to PAP, one problem is that conjugated PAP and unconjugated PAP are so similar in size that separation techniques can not distinguish between them.
Natural (also referred to as “native”) PAP is isolated from the pokeweed plant, and while attempts have been made to utilize natural PAP in a compound-toxin conjugate, such attempts have not proved reliable. As would be expected, variability in isoforms, from year to year and batch to batch, proved onerous and unworkable in the context of pharmaceutical quality control. Moreover, some isoforms did not conjugate, and different isoforms conjugated differently from each other.
Ideally, recombinant expression would provide control over these variables. Recombinant expression of PAP, however, has also met with difficulty. Previous expression in E. coli resulted in toxicity and inhibition of growth, as well as accumulation of recombinant pokeweed antiviral protein (rPAP) in inclusion bodies. In this regard, recombinant PAP required a separate solubilization step and subsequent refolding of the protein, resulting in poor yield and difficult scale-up. Other attempts in E. coli, S. cerevisiae, plants and P. pastoris resulted in low yields, or, in the case of P. pastoris, introduction of sequences that could potentially induce an inflammatory response. Moreover, recombinant PAP-compound fusion proteins either failed to bind or direct toxin to the target cells, or showed greatly reduced activity compared to natural PAP.
Therefore, a rPAP molecules having a free cysteine, conjugates made from them, and methods to produce rPAP, especially one that is high yield, results in easily folded and purified rPAP, and optionally provides an rPAP chemically available for conjugation, is a significant contribution.